Project Summary Arginine methylation is a common posttranslational modification (PTM), which is catalyzed by a family of nine arginine methyltransferases - the PRMTs. This PTM has been clearly implicated in a number of cellular processes, including transcriptional regulation. In the context of transcriptional regulation, PRMT4/CARM1 is a coactivator. The mechanisms behind its coactivator functions are unclear. In this grant proposal, we wil follow-up on two fundamental discoveries that we made in the past funding cycle: 1) that TDRD3 is an effector molecule for CARM1 methylation marks; and 2) that the H3R17me2a mark, which is deposited by CARM1, is dynamic in nature and thus suggests the existence of a demethylase that counteracts CARM1 activity. Our working hypothesis for this proposal is that CARM1 methylation of histone tails in a dynamic fashion, promotes the recruitment of the TDRD3 protein complex, which in-turn helps resolve R-loops that form at heavily transcribed genes. With the successful completion of the proposed studies, we wil have established the mechanism of how CARM1 functions as a transcriptional coactivator. Furthermore, we hope to identify the elusive arginine demethylase that counteracts the coactivator functions of CARM1. We propose that the CARM1?TDRD3?TOP3B axis plays a major role in regulating transcription, especially at heavily transcribed loci. We envision that CARM1 is recruited to promoters by transcription factors. There it methylates a number of different proteins, including histone H3 and RNAPII. These methylation events are read by TDRD3, which in turn recruits TOP3B to these actively transcribed genes. The TDRD3/TOP3B complex will thus be concentrated between the transcriptional start site and the elongating RNAPII. This is a region that is negatively supercoiled and prone to R-loop formation. It is known that TOP3B targets negatively supercoiled DNA. Thus, this topoisomerase is delivered to, and concentrated at, its potential substrates by CARM1 activity and TDRD3. By reducing negative supercoiling and resolving R-loop, TOP3B will promote transcription. TDRD3 has been identified as one of a small set of candidate genes used for a prognosis scoring system of breast cancer. Higher expression of TDRD3 is linked to a poor prognosis for breast cancer patient survival. TOP3B was also found to be overexpressed in breast ductal carcinomas, and strong associations with reduced patient survival and increased metastasis. Importantly, CARM1 levels are also elevated in aggressive breast tumors. Thus, there is clear evidence that the CARM1?TDRD3?TOP3B axis is disregulated, particularly in breast cancer. This axis is currently being targeted for drug development.